Apoptosis plays a variety of important roles in the immune system, and it is the goal of this application to elucidate the role of Bcl-2 family members in cell death. A critical decision point in the regulation of apoptosis occurs when the mitochondrial outer membrane permeabilizes to release proteins from the intermembrane space. The Bcl-2 family of proteins are thought to play a key role in the regulation of mitochondrial membrane permeabilization. Three fundamental aspects of the regulation of the Bcl-2 family of proteins will be addressed. In the first aim, we will examine the transcriptional regulation of the pro-apoptotic Bcl-2 family members during and after apoptotic stimulation (UV Irradiation, p53-induced, DNA damage, ER stress, activation induced cell death, and cytoskeletal disruption). This will be achieved through the use of RT real-time PCR techniques that have been established in the laboratory. In the second aim, we will examine the activation state of pro-apoptotic Bcl-2 family members in response to various apoptotic stimuli. This will be achieved through the use of a novel "Bcl-xL trap" that will interact with activated pro-apoptotic Bcl-2 family members. In the third aim, we will extend our studies to determine which pro-apoptotic Bcl-2 family members are necessary, sufficient, or redundant for induction of apoptosis. This will be achieved through the use of BiRNA. Additionally, we will use a call free apoptotic assay to determine which pro-apoptotic Bcl-2 family members have the ability to induce the release of cytochrome-c. The research outlined in this application focuses on the interaction between the pro-apoptotic and anti-apoptotic Bcl-2 family members in response to apoptotic stimulation. Our studies will elucidate those Bcl-2 family members that are: 1) expressed and/or upregulated, 2) activated, 3) necessary, sufficient, or redundant for induction of apoptosis.